The SaPI master repressor and the bacteriophage Duts: a tale of parasitism, evolution, and interspecific transfer

Staphylococcus aureus pathogenicity islands (SaPIs) form part of the wider family of phage inducible chromosomal islands (PICIs), which are extremely mobile phage satellites. SaPIs are clinically relevant, encoding and disseminating virulence and fitness factors to promote bacterial evolution and pathogenesis. These islands are innately linked to their helper phage’s life cycle, requiring phage-mediated induction and packaging for transfer between bacterial hosts. The inherent association between SaPIs and helper phages is one of the best characterised examples of virus satellite interactions in prokaryotic cells. The SaPIs sit passively in the host chromosome, controlled by the SaPI-encoded master repressor protein; the Stl. Interaction between the SaPIs and their helper phages begins when a phage-encoded protein binds to the Stl to de-repress the island. Different SaPIs encode different Stl repressors, which require diverse phage-encoded proteins for derepression. The best characterised phage-encoded inducers are the trimeric dUTPases (Duts), which traditionally prevent the misincorporation of uracil into DNA, but have been proposed as regulatory proteins that induce SaPIbov1. Recently, the structurally dissimilar phage-encoded dimeric Dut of NM1 has also been shown to mobilize SaPIbov1, but how this is accomplished remains unsolved. Here, this work expands the family of SaPIbov1 phage-encoded inducers to include the subset of phage-encoded dimeric Duts. This develops upon the theory that specific phage-proteins are required for SaPI induction, broadening this vision to show that SaPIs instead target conserved phage mechanisms. Using SaPIbov1 as an example, this study indicates the StlSaPIbov1 can interact with the structurally distinct dimeric and trimeric Duts, whose only shared value is their role for the phage. Furthermore, this study highlights that, although these two proteins are completely unrelated, they both encode one highly variable region (named motif VI). Indeed, the mechanism for the dimeric Dut interaction with the StlSaPIbov1 is investigated here and a number of parallels to the trimeric Dut mode of action are apparent. These results indicate that the structurally divergent dimeric and trimeric Duts interact with the SaPIbov1 Stl using analogous, but distinct mechanisms that represent a fascinating example of convergent evolution. Furthermore, investigations into the SaPIbov1 Stl indicate that Stl accomplishes these interactions with the dimeric and trimeric Duts by using separate domains for each interaction. This intriguing Stl evolution is an elegant strategy utilised by the SaPIs to overcome phage-encoded inducer mutation or exchange. This highlights the continual arms race between the phages, which aim to escape SaPI induction, and the SaPIs, which must overcome these changes for island induction to occur. Furthermore, these results highlight the potential for Stl targeting of structurally diverse, functionally related phage proteins to facilitate intra- and inter-specific transfer. Overall, these results highlight the SaPIs as a fascinating subcellular parasite of the phages, which have evolved a novel strategy to target their phage inducer proteins and achieve transfer. Likewise, this study shows the biological significance of the dimeric Duts as regulatory molecules

Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity island induction involves novel players in the party

13 páginas, 5 figuras, 3 tablas, material suplementario en NAR onlineWe have recently proposed that the trimeric staphylococcal phage encoded dUTPases (Duts) are signaling molecules that act analogously to eukaryotic G-proteins, using dUTP as a second messenger. To perform this regulatory role, the Duts require their characteristic extra motif VI, present in all the staphylococcal phage coded trimeric Duts, as well as the strongly conserved Dut motif V. Recently, however, an alternative model involving Duts in the transfer of the staphylococcal islands (SaPIs) has been suggested, questioning the implication of motifs V and VI. Here, using state-of the-art techniques, we have revisited the proposed models. Our results confirm that the mechanism by which the Duts derepress the SaPI cycle depends on dUTP and involves both motifs V and VI, as we have previously proposed. Surprisingly, the conserved Dut motif IV is also implicated in SaPI derepression. However, and in agreement with the proposed alternative model, the dUTP inhibits rather than inducing the process, as we had initially proposed. In summary, our results clarify, validate and establish the mechanism by which the Duts perform regulatory functions.Ministerio de Economia y Competitividad (Spain) [BIO2013-42619-P to A.M.]; Medical Research Council (UK) [MR/M003876/1]; ERC-ADG-2014 Dut-signal (from EU) [Proposal no 670932 to J.R.P]; CSIC JAE-Doc Postdoctoral contract (Programa «Junta para la Ampliación de Estudios»), European Social Fund (to E.M.); FPU13/02880 (to J.R.C.), FPI BES-2014-068617 Predoctoral Fellowships (to C.A.). Diamond Light Source block allocation group (BAG) Proposal [MX10121]; Spanish Synchrotron Radiation Facility ALBA Proposal [2014060897]; European Community's Seventh Framework Programme [FP7/2007-2013]; BioStruct-X [283570]. Funding for open access charge: Ministerio de Economia y Competitividad (Spain) [BIO2013-42619-P to A.M.]; Medical Research Council (UK) [MR/M003876/1]; ERC-ADG-2014 Dut-signal (from EU) [Proposal no 670932 to J.R.P].Peer reviewe

Screening for Highly Transduced Genes in Staphylococcus aureus Revealed Both Lateral and Specialized Transduction.

Bacteriophage-mediated transduction of bacterial DNA is a major route of horizontal gene transfer in the human pathogen, Staphylococcus aureus. Transduction involves the packaging of bacterial DNA by viruses and enables the transmission of virulence and resistance genes between cells. To learn more about transduction in S. aureus, we searched a transposon mutant library for genes and mutations that enhanced transfer mediated by the temperate phage, ϕ11. Using a novel screening strategy, we performed multiple rounds of transduction of transposon mutant pools selecting for an antibiotic resistance marker within the transposon element. When determining the locations of transferred mutations, we found that the screen had selected for just 1 or 2 transposon mutant(s) within each pool of 96 mutants. Subsequent analysis showed that the position of the transposon, rather than the inactivation of bacterial genes, was responsible for the phenotype. Interestingly, from multiple rounds, we identified a pattern of transduction that encompassed mobile genetic elements as well as chromosomal regions both upstream and downstream of the phage integration site. The latter was confirmed by DNA sequencing of purified phage lysates. Importantly, transduction frequencies were lower for phage lysates obtained by phage infection rather than induction. Our results confirmed previous reports of lateral transduction of bacterial DNA downstream of the integrated phage but also indicated a novel form of specialized transduction of DNA upstream of the phage. These findings illustrated the complexity of transduction processes and increased our understanding of the mechanisms by which phages transfer bacterial DNA. IMPORTANCE Horizontal transfer of DNA between bacterial cells contributes to the spread of virulence and antibiotic resistance genes in human pathogens. For Staphylococcus aureus, bacterial viruses play a major role in facilitating the horizontal transfer. These viruses, termed bacteriophages, can transfer bacterial DNA between cells by a process known as transduction, which despite its importance is only poorly characterized. Here, we employed a transposon mutant library to investigate transduction in S. aureus. We showed that the genomic location of bacterial DNA relative to where bacteriophages integrated into that bacterial genome affected how frequently that DNA was transduced. Based on serial transduction of transposon mutant pools and direct sequencing of bacterial DNA in bacteriophage particles, we demonstrated both lateral and specialized transduction. The use of mutant libraries to investigate the genomic patterns of bacterial DNA transferred between cells could help us understand how horizontal transfer influences virulence and resistance development

The structure of a polygamous repressor reveals how phage-inducible chromosomal islands spread in nature

Stl is a master repressor encoded by Staphylococcus aureus pathogenicity islands (SaPIs) that maintains integration of these elements in the bacterial chromosome. After infection or induction of a resident helper phage, SaPIs are de-repressed by specific interactions of phage proteins with Stl. SaPIs have evolved a fascinating mechanism to ensure their promiscuous transfer by targeting structurally unrelated proteins performing identically conserved functions for the phage. Here we decipher the molecular mechanism of this elegant strategy by determining the structure of SaPIbov1 Stl alone and in complex with two structurally unrelated dUTPases from different S. aureus phages. Remarkably, SaPIbov1 Stl has evolved different domains implicated in DNA and partner recognition specificity. This work presents the solved structure of a SaPI repressor protein and the discovery of a modular repressor that acquires multispecificity through domain recruiting. Our results establish the mechanism that allows widespread dissemination of SaPIs in nature

A fascinating example of convergent evolution involves Staphylococcus aureus phage encoded dimeric and trimeric dUTPases in signaling

Póster presentado al XL SEBBM Congress. Barcelona, 23-26 de octubre de 2017The dUTPase (Dut) enzymes prevent the misincorporation of uracil into the DNA and are encoded by almost all free-living organisms and some viruses. We have previously showed that phage-encoded trimeric Duts mediates the Staphylococcus aureus pathogenicity island (SaPIs) transfer by interacting to the SaPI-encoded repressor Stl, proposing that these Duts are regulatory proteins. Some S. aureus phages encode structurally unrelated dimeric Duts instead trimeric Duts. Surprisingly, a recent work, has involved one of these predicted dimeric Duts in the transfer of SaPIs by interacting with the same Stl repressor. With the aim of decipher the molecular basis of SaPI induction by dimeric Duts and to compare with the mechanism reported for trimeric Duts, we examined the SaPI mobilization capacity of dimeric Duts in vivo as well as its binding capacity to Stl repressor in vitro. We analyzed the effect of the dUTP in the Stl-Dut interaction, and finally, we solved the 3-D structure by X-ray crystallography of several dimeric Duts in different activation states. Our results show that dimeric and trimeric Duts from S. aureus phages present a striking parallelism in the mechanism of SaPI mobilization. These similarities would confirm the role of dUTP as new nucleotide with second messenger function. However, some differences suggest peculiarities in the molecular mechanism of Stl recognition and binding for each type of Duts. Our results support the idea that the signalling role of the Dut proteins is an important force driving evolution and speciation.J.Rafael Ciges-Tomas. PhD student, fellowship FPU13/02880 awarded by Ministry of Education, Culture and SportPeer reviewe

Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity island induction involves novel players in the party

We have recently proposed that the trimeric staphylococcal phage encoded dUTPases (Duts) are signaling molecules that act analogously to eukaryotic G-proteins, using dUTP as a second messenger. To perform this regulatory role, the Duts require their characteristic extra motif VI, present in all the staphylococcal phage coded trimeric Duts, as well as the strongly conserved Dut motif V. Recently, however, an alternative model involving Duts in the transfer of the staphylococcal islands (SaPIs) has been suggested, questioning the implication of motifs V and VI. Here, using state-of the-art techniques, we have revisited the proposed models. Our results confirm that the mechanism by which the Duts derepress the SaPI cycle depends on dUTP and involves both motifs V and VI, as we have previously proposed. Surprisingly, the conserved Dut motif IV is also implicated in SaPI derepression. However, and in agreement with the proposed alternative model, the dUTP inhibits rather than inducing the process, as we had initially proposed. In summary, our results clarify, validate and establish the mechanism by which the Duts perform regulatory functions

Cross-species communication via agr controls phage susceptibility in Staphylococcus aureus

Summary: Bacteria use quorum sensing (QS) to coordinate group behavior in response to cell density, and some bacterial viruses (phages) also respond to QS. In Staphylococcus aureus, the agr-encoded QS system relies on accumulation of auto-inducing cyclic peptides (AIPs). Other staphylococci also produce AIPs of which many inhibit S. aureus agr. We show that agr induction reduces expression of tarM, encoding a glycosyltransferase responsible for α-N-acetylglucosamine modification of the major S. aureus phage receptor, the wall teichoic acids. This allows lytic phage Stab20 and related phages to infect and kill S. aureus. However, in mixed communities, producers of inhibitory AIPs like S. haemolyticus, S. caprae, and S. pseudintermedius inhibit S. aureus agr, thereby impeding phage infection. Our results demonstrate that cross-species interactions dramatically impact phage susceptibility. These interactions likely influence microbial ecology and impact the efficacy of phages in medical and biotechnological applications such as phage therapy

Pirating conserved phage mechanisms promotes promiscuous staphylococcal pathogenicity island transfer

Targeting conserved and essential processes is a successful strategy to combat enemies. Remarkably, the clinically important Staphylococcus aureus pathogenicity islands (SaPIs) use this tactic to spread in nature. SaPIs reside passively in the host chromosome, under the control of the SaPI-encoded master repressor, Stl. It has been assumed that SaPI de-repression is effected by specific phage proteins that bind to Stl, initiating the SaPI cycle. Different SaPIs encode different Stl repressors, so each targets a specific phage protein for its de-repression. Broadening this narrow vision, we report here that SaPIs ensure their promiscuous transfer by targeting conserved phage mechanisms. This is accomplished because the SaPI Stl repressors have acquired different domains to interact with unrelated proteins, encoded by different phages, but in all cases performing the same conserved function. This elegant strategy allows intra-and inter-generic SaPI transfer, highlighting these elements as one of nature’s most fascinating subcellular parasites